Selective 5mC Labeling in Low-Input cfDNA via Enzymatic Conversion
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Solution Overview
Problem
Current methods for sequencing 5-methylcytosine (5mC) in circulating cell-free DNA (cfDNA) are limited by the need for large DNA amounts and inability to distinguish 5mC from 5-hydroxymethylcytosine (5hmC, preventing the decoupling of repressive 5mC and activating 5hmC signals.
Innovation Solution
A method involving selective chemical labeling, where a blocking group is added to endogenous 5hmC, converting 5mC to 5hmC, and attaching a linking group followed by an affinity tag for enrichment and sequencing, allowing for the generation of a library from low-input DNA samples, including cfDNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite sequencing is used for 5mC sequencing, then sequencing capability is achieved, but DNA degradation occurs and large amounts of DNA are required
Solution Approach 1:
The patent uses TET enzyme as an intermediary to convert 5mC to 5hmC, and glucose moiety as a mediator to selectively label 5hmC. This indirect labeling approach avoids the harsh bisulfite treatment that degrades DNA, enabling sequencing of low-input cfDNA samples while maintaining DNA integrity and reducing the amount of DNA required from nanogram to picogram levels.
Solution Approach 2:
The patent changes the chemical state of 5mC by converting it to 5hmC through TET enzyme treatment. This parameter change (oxidation state) enables selective labeling with glucose moieties that specifically recognize 5hmC, allowing for gentle, non-degradative enrichment of methylated DNA sequences from low-input samples.
2Measurement precision
If bisulfite sequencing is used, then 5mC detection is achieved, but 5mC and 5hmC cannot be distinguished
Solution Approach 1:
The patent applies preliminary blocking of endogenous 5hmC with glucose moieties before TET treatment. This preliminary action preserves the original 5hmC sites while converting 5mC to 5hmC, enabling subsequent differentiation between the two epigenetic marks through selective labeling and sequencing.
Solution Approach 2:
The patent uses TET enzyme as an intermediary to selectively convert 5mC to 5hmC after endogenous 5hmC has been blocked. This intermediary action enables the decoupling of 5mC and 5hmC signals, allowing both epigenetic marks to be detected and distinguished separately, preserving complete epigenetic information.
3Measurement precision
If conventional sequencing methods are used on cfDNA, then sequencing is achieved, but the low input amount of cfDNA limits application
Solution Approach 1:
The patent extracts and enriches only the 5mC-containing DNA sequences from the complex cfDNA mixture through selective chemical labeling and affinity purification. This extraction approach concentrates the target sequences from low-input samples, enabling accurate sequencing without requiring large amounts of total cfDNA.
Solution Approach 2:
The patent changes the chemical parameters of cfDNA by selectively oxidizing 5mC to 5hmC and labeling with glucose moieties. This parameter change enables specific enrichment of methylated sequences from picogram-level cfDNA inputs, overcoming the limitation of conventional methods that require nanogram to microgram amounts of DNA.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables the selective sequencing of 5mC from low-input DNA samples, decoupling 5mC and 5hmC signals, and provides a non-invasive approach for cancer diagnostics by identifying tissue-of-origin and distinguishing between open and closed chromatin states.
Implementation Method 1
converting the 5mC in the DNA sample to 5hmC
Implementation Method 2
enriching for the affinity tagged DNA from step (f) by affinity purification
Data Source
AI summary
The present disclosure provides methods for selectively tagging 5-methylcytosine in a DNA sample and using this approach for genome-wide profiling of 5-methylcytosine in a low input DNA sample such as circulating cell-free DNA.